Experimental cancer genetics

The Experimental cancer genetics team aims to understand the fundamental genetic mechanisms by which cancers develop.

Their approach to use large-scale DNA sequencing to identify candidate cancer genes and to study these genes in model
systems such as in mice and cells. Another aspect of their work is to perform genetic screens in models systems using
tools such as CRISPR and transposons. A particular interest is synthetic lethality, essential genes and immune
modulators.

Background

The genetic basis of cancer

Cancer occurs when there is an accumulation of genetic damage that confers a selective advantage on a cell, allowing
it to evade normal growth control processes. Uncontrolled growth and reproduction of these damaged cells ultimately
results in tumour formation. While some of the key events at the molecular level that are involved in cancer
formation are known, there is still much work to be done to identify other genetic changes important in cancer
formation that could represent new diagnostic markers or targets for new anti-cancer medicines.

The mutational landscapes of genetic and chemical models of Kras-driven lung cancer.

1] Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158, USA [2] Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94158, USA.

Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.

Research

High-throughput sequencing efforts are uncovering the mutation profiles associated to cancer in a comprehensive and unbiased fashion. Nevertheless, further analyses of the identified alterations are necessary to highlight those genes implicated in disease development, as well as, those with therapeutical potential. Recently, we generated a list of candidate genes commonly truncated based on the analysis of >7500 cancer samples across 28 tissues. Therefore, we are generating a panel of isogenic iPS cell lines to perform high-throughput genetic and drug screenings. Overall, this study will identify molecular interactions of the selected cancer-related genes thus revealing potential new roles and candidate therapies.

Background &amp; aims: Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors.

Methods: By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1-PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort.

Results: Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1-PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC.

Conclusions: In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1-PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time.

Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

Background &amp; aims: The Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer.

Methods: The intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This was achieved through Cre expression under the control of an albumin and α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain [NICD]). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A Notch gene signature was generated using the nearest template prediction method.

Results: AFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2; these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of insulin-like growth factor signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of mastermind-like 1 reduced their proliferation in vitro.

Conclusions: Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.

Purpose: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer.

Background &amp; aims: IGF signaling has a relevant role in a variety of human malignancies. We analyzed the underlying molecular mechanisms of IGF signaling activation in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and assessed novel targeted therapies blocking this pathway.

Methods: An integrative molecular dissection of the axis was conducted in a cohort of 104 HCCs analyzing gene and miRNA expression, structural aberrations, and protein activation. The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro and in a xenograft model of HCC.

Results: Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters P3 and P4-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with mTOR signaling (p=0.035) and was clearly enriched in the Proliferation subclass of the molecular classification of HCC (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 delayed tumor growth and prolonged survival, reducing proliferation rates and inducing apoptosis.

Conclusions: Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.

Mount Sinai Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.

Background &amp; aims: The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway.

Conclusions: MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

Martin Del Castillo Velasco Herrera

- PhD Student

I obtained my Bachelor degree in Genome Sciences (Hons) from the National Autonomous University of Mexico in 2011. Prior to joining Sanger as a PhD Student, I participated in projects that focused in cancer gene discovery and high throughput sequencing data analysis.

Research

My PhD project focuses on the identification of genes involved in the metastatic process in melanoma. We use whole genome and transcriptome sequencing to identify potential candidate genes, followed by experimental validation using CRISPR-Cas9 experiments in-vitro and subsequently in-vivo murine models.

The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity.

Telomere-regulating genes and the telomere interactome in familial cancers.

Telomeres are repetitive sequence structures at the ends of linear chromosomes that consist of double-stranded DNA repeats followed by a short single-stranded DNA protrusion. Telomeres need to be replicated in each cell cycle and protected from DNA-processing enzymes, tasks that cells execute using specialized protein complexes such as telomerase (that includes TERT), which aids in telomere maintenance and replication, and the shelterin complex, which protects chromosome ends. These complexes are also able to interact with a variety of other proteins, referred to as the telomere interactome, to fulfill their biological functions and control signaling cascades originating from telomeres. Given their essential role in genomic maintenance and cell-cycle control, germline mutations in telomere-regulating proteins and their interacting partners have been found to underlie a variety of diseases and cancer-predisposition syndromes. These syndromes can be characterized by progressively shortening telomeres, in which carriers can present with organ failure due to stem cell senescence among other characteristics, or can also present with long or unprotected telomeres, providing an alternative route for cancer formation. This review summarizes the critical roles that telomere-regulating proteins play in cell-cycle control and cell fate and explores the current knowledge on different cancer-predisposing conditions that have been linked to germline defects in these proteins and their interacting partners.

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity.

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm) embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm) embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

Stefan Dentro

- PhD Student

Previously I've obtained a B.Sc. and M.Sc. in computer science, specialising in bioinformatics from Delft University of Technology in The Netherlands. For my masters thesis I've developed a classification approach that predicts the type of cancer through somatic point mutations.

Research

My project revolves around developing methods to construct the clonal architecture of tumours from sequencing data and applying these methods to available data sets. I'm part of ICGC-Pan-cancer where, together with the tumour heterogeneity and evolution working group, I work on uncovering the evolutionary story of 2500 tumours.

Richard Gunning

- PhD Student

BSc. Biochemistry at King's College London
MSc. Bioinformatics at King's College London
MRes. BBSRC DTP at Cambridge University

Research

Studying the Epigenetic regulation of Splicing in C57BL/6J mice

Marco Ranzani

- Postdoctoral Fellow

• July 2012- August 2013: Postdoctoral fellow at HSR-TIGET under the mentorship of Dr. E Montini.
• January 2008-July 2012: Ph.D. student under the supervision of Dr. E Montini, Prof. L Naldini and Prof. M van Lohuizen. Ph.D. program in Cellular and Molecular Biology by San Raffaele Vita-Salute University, Milan, Italy and The Open University, London, UK.
• September 2006-December 2007: Graduate fellow at HSR-TIGET under the supervision of Prof. L Naldini.
• July 2006: MSc degree in Medical Biotechnology and Molecular Medicine. University of Milan, Italy.
• July 2004: BSc in Medical Biotechnology. University of Milan, Italy.

Research

The aim of my project is to identify new therapies for the treatment of melanoma.
I am characterizing the sensitivity to a library of anticancer drugs and drug combinations of a collection of melanoma cell lines. I will combine these results with mutations, copy number variations and gene expression of each cell line to identify markers of drug resistance.
I will then utilized this knowledge to design new drug combinations for the efficient eradication of melanoma that will be validated in vitro and in vivo.

The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.

Molecular therapy : the journal of the American Society of Gene Therapy2014;22;12;2056-68

Cancer gene discovery goes mobile.

A new study describes a tool, Lentihop, for somatic insertional mutagenesis in human cells and uses this system in combination with cancer genome data to define new genes and pathways involved in sarcoma development. Gene discovery in this way suggests that we are far from a complete catalog of cancer drivers.

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

Molecular therapy : the journal of the American Society of Gene Therapy2014;22;4;774-85

Cancer gene discovery: exploiting insertional mutagenesis.

Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future.

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

Tumour cells often express deregulated profiles of chemokine receptors that regulate cancer cell migration and proliferation. Notch1 pathway activation is seen in T cell acute lymphoblastic leukaemia (T-ALL) due to the high frequency of Notch1 mutations affecting approximately 60% of patients, causing ligand-independent signalling and/or prolonging Notch1 half-life. We have investigated the possible regulative role of Notch1 on the expression and function of chemokine receptors CCR5, CCR9 and CXCR4 that play a role in determining blast malignant properties and localization of extramedullary infiltrations in leukaemia. We inhibited the pathway through γ-Secretase inhibitor and Notch1 RNA interference and analysed the effect on the expression and function of chemokine receptors. Our results indicate that γ-Secretase inhibitor negatively regulates the transcription level of the CC chemokine receptors 5 and 9 in T-ALL cell lines and patients' primary leukaemia cells, leaving CXCR4 expression unaltered. The Notch pathway also controls CCR5- and CCR9-mediated biological effects, ie chemotaxis and proliferation. Furthermore, engaging CCR9 through CCL25 administration rescues proliferation inhibition associated with abrogation of Notch activity. Finally, through RNA interference we demonstrated that the oncogenic isoform in T-ALL, Notch1, plays a role in controlling CCR5 and CCR9 expression and functions. These findings suggest that Notch1, acting in concert with chemokine receptors pathways, may provide leukaemia cells with proliferative advantage and specific chemotactic abilities, therefore influencing tumour cell progression and localization.

A recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γ-retroviral vector-based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection.

gamma-Retroviral vectors (gammaRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gammaRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gammaRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gammaRVs. This integration-site bias was intrinsic to gammaRVs, as it was also observed for SIN gammaRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Daniela Robles Espinoza

- Postdoctoral Fellow

I graduated with a Bachelor degree in Genome Sciences from the National Autonomous University of Mexico in 2009. I then worked for a year in two research groups focusing in cancer gene discovery and signaling networks, before joining the Sanger Institute as a PhD student.

Research

My PhD project and current work focus on the identification of novel melanoma susceptibility genes in predisposed families, and the mechanisms by which they might cause disease. We utilise whole- and targeted- exome sequencing and bioinformatic tools followed by the biological validation of targets in an effort to understand the genetics underlying this disease.

References

Nonsense mutations in the shelterin complex genes ACD and TERF2IP in familial melanoma.

Background: The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families.

Results: Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma.

Conclusions: Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility.

Telomere-regulating genes and the telomere interactome in familial cancers.

Telomeres are repetitive sequence structures at the ends of linear chromosomes that consist of double-stranded DNA repeats followed by a short single-stranded DNA protrusion. Telomeres need to be replicated in each cell cycle and protected from DNA-processing enzymes, tasks that cells execute using specialized protein complexes such as telomerase (that includes TERT), which aids in telomere maintenance and replication, and the shelterin complex, which protects chromosome ends. These complexes are also able to interact with a variety of other proteins, referred to as the telomere interactome, to fulfill their biological functions and control signaling cascades originating from telomeres. Given their essential role in genomic maintenance and cell-cycle control, germline mutations in telomere-regulating proteins and their interacting partners have been found to underlie a variety of diseases and cancer-predisposition syndromes. These syndromes can be characterized by progressively shortening telomeres, in which carriers can present with organ failure due to stem cell senescence among other characteristics, or can also present with long or unprotected telomeres, providing an alternative route for cancer formation. This review summarizes the critical roles that telomere-regulating proteins play in cell-cycle control and cell fate and explores the current knowledge on different cancer-predisposing conditions that have been linked to germline defects in these proteins and their interacting partners.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

Cross-species analysis of mouse and human cancer genomes.

Fundamental advances in our understanding of the human cancer genome have been made over the last five years, driven largely by the development of next-generation sequencing (NGS) technologies. Here we will discuss the tools and technologies that have been used to profile human tumors, how they may be applied to the analysis of the mouse cancer genome, and the results thus far. In addition to mutations that disrupt cancer genes, NGS is also being applied to the analysis of the transcriptome of cancers, and, through the use of techniques such as ChIP-Seq, the protein-DNA landscape is also being revealed. Gaining a comprehensive picture of the mouse cancer genome, at the DNA level and through the analysis of the transcriptome and regulatory landscape, will allow us to "biofilter" for driver genes in more complex human cancers and represents a critical test to determine which mouse cancer models are faithful genetic surrogates of the human disease.

Cake: a bioinformatics pipeline for the integrated analysis of somatic variants in cancer genomes.

We have developed Cake, a bioinformatics software pipeline that integrates four publicly available somatic variant-calling algorithms to identify single nucleotide variants with higher sensitivity and accuracy than any one algorithm alone. Cake can be run on a high-performance computer cluster or used as a stand-alone application. Availabilty: Cake is open-source and is available from http://cakesomatic.sourceforge.net/

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity.

Marcela Sjoberg

- Postdoctoral Fellow

I am a Molecular Biotechnology Engineer (2002), with a PhD degree in Molecular, Cellular Biology and Neuroscience (2006) from Universidad de Chile, Santiago, Chile. I did my postdoctoral training at the MRC Clinical Sciences Centre (Imperial College London, UK) examining mechanisms that control genome function in stem cells and immune cells during differentiation and cell cycle (2006-2010). Afterwards I acted as a consultant for the Chilean Stem Cell bank VidaCel®. In 2012 I joined the Sanger Institute as a Postdoctoral Fellow to develop a work package of the BLUEPRINT consortium project, aiming at generating epigenomic maps of blood cell types.

Research

Different cell types arise from the acquisition and maintenance of chemical modifications that modulate their genome function and define their epigenome. Cancer cells loose their identity and display significant alterations in their epigenome, however how genomic and epigenomic changes contribute to cancer development needs further examination. I am using strain-specific genome variation to uncover epigenome and gene expression changes in blood cells. By employing high-throughput sequencing technologies I have generated epigenomic (ChIP-seq and WGBS-oxWGBS) and transcriptomic (RNA-seq) profiles and we are currently cataloguing and quantifying genotype mediated epigenome variations, with a specific focus on DNA methylation and histone modifications.

Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

Reversible cellular quiescence is critical for developmental processes in metazoan organisms and is characterized by a reduction in cell size and transcriptional activity. We show that the Aurora B kinase and the polycomb protein Ring1B have essential roles in regulating transcriptionally active genes in quiescent lymphocytes. Ring1B and Aurora B bind to a wide range of active promoters in resting B and T cells. Conditional knockout of either protein results in reduced transcription and binding of RNA Pol II to promoter regions and decreased cell viability. Aurora B phosphorylates histone H3S28 at active promoters in resting B cells as well as inhibiting Ring1B-mediated ubiquitination of histone H2A and enhancing binding and activity of the USP16 deubiquitinase at transcribed genes. Our results identify a mechanism for regulating transcription in quiescent cells that has implications for epigenetic regulation of the choice between proliferation and quiescence.

Alzheimer's disease (AD) is characterized by the accumulation of protein filaments, namely extracellular amyloid-beta (Abeta) fibrils and intracellular neurofibrillary tangles, which are composed of aggregated hyperphosphorylated tau. Tau hyperphosphorylation is the product of deregulated Ser/Thr kinases such as cdk5 and GSK3beta. In addition, tau hyperphosphorylation also occurs at Tyr residues. To find a link between Abeta and tau phosphorylation, we investigated the effects of short-term Abeta treatments on SHSY-5Y cells. We analyzed phosphorylated tau variants in lipid rafts and the possible role of Tyr18 and Ser396/404 tau phosphorylation in Abeta-induced signaling cascades. After 2 min of Abeta treatment, phospho-Tyr18-tau and its association with rafts increased. Phospho-Ser 396/404-tau became detectable in rafts after 10 min treatment, which temporally correlated with the detection of cdk5 and p35 activator in lipid rafts. To determine the role of cdk5 in tau phosphorylation at Ser396/404 in lipid rafts, we pre-incubated cells with cdk5 inhibitor roscovitine, and observed that the Abeta-induced tau phosphorylation at Ser 396/404 in rafts was abolished as well as cdk5/p35 association with rafts. These data suggest a role for cdk5 in the Abeta-promoted early events involving tau hyperphosphorylation, and their possible implications for AD pathogenesis.

Mutations in the GH receptor gene have been identified as the cause of growth hormone insensitivity syndrome (GHIS), a rare autosomal recessive disorder. We studied the clinical and biochemical characteristics and the coding sequence and intron-exon boundaries of the GH receptor gene in a consanguineous family with severe short stature which consisted of two patients, their parents and five siblings. The two adolescents had heights of -4.7 and -5.5 SDS, respectively, with elevated growth hormone associated with low IGF-I, IGFBP-3 and GHBP concentrations. Molecular analysis of the GH receptor gene revealed a mutation in exon 6, present in both patients This mutation, E180 splice, has been previously described in an Ecuadorian cohort, and in one Israeli and six Brazilian patients. We determined the GH receptor haplotypes based on six polymorphic sites in intron 9. Co-segregation of the E180splice mutation with haplotype I was found in this family, compatible with a common Mediterranean ancestor, as shown for previous cases with the E180splice mutation described to date.

Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1beta away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression.

The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. A nuclear form of tau has been described in neuronal and non-neuronal cells, which displays a nucleolar localization during interphase but is associated with nucleolar-organizing regions in mitotic cells. In the present study, based on immunofluorescence, immuno-FISH and confocal microscopy, we show that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-rich alpha-satellite DNA sequences organized as constitutive heterochromatin. By using gel retardation, we demonstrate that tau not only colocalizes with, but also specifically binds to, AT-rich satellite DNA sequences apparently through the recognition of AT-rich DNA stretches. Here we propose a functional role for nuclear tau in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes. Since nuclear tau has also been found in neurons from patients with Alzheimer's disease (AD), aberrant nuclear tau could affect the nucleolar organization during the course of AD. We discuss nucleolar tau associated with AT-rich alpha-satellite DNA sequences as a potential molecular link between trisomy 21 and AD.

Alzheimer's disease is the principal cause of dementia throughout the world and the fourth cause of death in developed economies.This brain disorder is characterized by the formation of brain protein aggregates, namely, the paired helical filaments and senile plaques. Oxidative stress during life, neuroinflamamtion, and alterations in neuron-glia interaction patterns have been also involved in the etiopathogenesis of this disease. In recent years, cumulative evidence has been gained on the involvement of alteration in neuronal lipoproteins activity, as well as on the role of cholesterol and other lipids in the pathogenesis of this neurodegenerative disorder. In this review, we analyze the links between changes in cholesterol homeostasis, and the changes of lipids of major importance for neuronal activity and Alheimer's disease. The investigation on the fine molecular mechanisms underlying the lipids influence in the etiopathogenesis of Alzheimer's disease may shed light into its treatment and medical management.

Study of GH sensitivity in chilean patients with idiopathic short stature.

Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Santiago.

We hypothesized that some children with idiopathic short stature in Chile might bear heterozygous mutations of the GH receptor. We selected 26 patients (3 females, 23 males) from 112 patients who consulted for idiopathic short stature at the University of Chile. Their chronological age was 8.3 +/- 1.9, and bone age was 6.1 +/- 1.0 yr. Their height was -3.0 +/- 0.7 SDS; IGF-I, -1.2 +/- 1.1 SD; IGF binding protein 3, -0.7 +/- 2.0 SDS; and GH binding protein, 0.4 +/- 0.8 SDS. Patients were admitted, and blood samples were obtained every 20 min to determine GH concentrations overnight. Coding sequences and intron-exon boundaries of exons 2-10 of GH receptor gene were amplified by PCR and subsequently analyzed through single-strand conformational analysis. Mean serum GH concentration, over 12-h, was 0.20 +/- 0.08 nM; pulse amplitude, 0.40 +/- 0.15 nM; number of peaks, 5.8 +/-1.5 peaks/12 h; peak value of GH during the 12-h sampling, 1.03 +/- 0.53 nM; and area under the curve, 151.4 +/- 56.1 nM/12 h. There were positive correlations between mean GH vs. area under the curve (P < 0.001) and GH peak (P < 0.01). The single-strand conformational analysis of the GH receptor gene showed abnormal migration for exon 6 in 9 patients and for exon 10 in 9 patients, which (by sequence analysis) corresponded to 2 polymorphisms of the GH receptor gene: an A-to-G transition in third position of codon 168 in exon 6 and a C-to-A transversion in the first position of codon 526 in exon 10. We further sequenced all coding exons and intron-exon boundaries in the most affected patients (nos. 6, 9, 11, 14, 15, 16, and 23). This analysis revealed a C-to-T transition in codon 161 of exon 6 in patient 23, which results in an amino acid change (Arg to Cys) in an heterozygous form in the patient and his father. In conclusion, the results of our study suggest that, in Chilean patients with idiopathic short stature, GH receptor gene mutations are uncommon, although we cannot exclude mutations that were missed by single-strand conformational analysis or mutations within introns or in the promoter regions of the GH receptor gene.

Louise Van Der Weyden

- Senior Staff Scientist

Dr. van der Weyden received her Ph.D. in cancer biology from the University of Sydney, Australia, before moving to the UK to work as a Post-doctoral Research Fellow at the WTSI. In Professor Allan Bradley’s laboratory her research focused on generating and characterising mouse models of cancer. This was further built on in Dr. David Adams’ laboratory where she used insertional mutagenesis (retroviruses and transposons) to identify genes that co-operate in tumorigenesis. Most recently, she has undertaken a research program that focuses on understanding the nature of metastasis, in particular melanoma metastasis.

Research

Most cancer patients die from metastasis rather than the primary tumour. This is what makes metastasis such an intriguing and important area of research. Melanoma is known for its intense metastatic propensity, so is a good model to study. I'm sequencing mouse melanoma cell lines with differing metastatic capacities and using cross-species comparison to identify which differentially expressed or mutated genes are also found in human datasets. I'm also performing an ‘experimental metastasis assay’ on KO mouse lines coming through the Mouse Genetics Program at the WTSI to identify mutants that show altered levels of pulmonary metastasis relative to controls.

Department of Haematology, University of Cambridge, and National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom;

NBEAL2 encodes a multidomain scaffolding protein with a putative role in granule ontogeny in human platelets. Mutations in NBEAL2 underlie gray platelet syndrome (GPS), a rare inherited bleeding disorder characterized by a lack of α-granules within blood platelets and progressive bone marrow fibrosis. We present here a novel Nbeal2(-/-) murine model of GPS and demonstrate that the lack of α-granules is due to their loss from platelets/mature megakaryocytes (MKs), and not by initial impaired formation. We show that the lack of Nbeal2 confers a proinflammatory phenotype to the bone marrow MKs, which in combination with the loss of proteins from α-granules drives the development of bone marrow fibrosis. In addition, we demonstrate that α-granule deficiency impairs platelet function beyond their purely hemostatic role and that Nbeal2 deficiency has a protective effect against cancer metastasis.

Cancer gene discovery goes mobile.

A new study describes a tool, Lentihop, for somatic insertional mutagenesis in human cells and uses this system in combination with cancer genome data to define new genes and pathways involved in sarcoma development. Gene discovery in this way suggests that we are far from a complete catalog of cancer drivers.

Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary Medicine and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a 'progression network' that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.

Cancer of mice and men: old twists and new tails.

In this review we set out to celebrate the contribution that mouse models of human cancer have made to our understanding of the fundamental mechanisms driving tumourigenesis. We take the opportunity to look forward to how the mouse will be used to model cancer and the tools and technologies that will be applied, and indulge in looking back at the key advances the mouse has made possible.

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity.

Using mice to unveil the genetics of cancer resistance.

In the UK, four in ten people will develop some form of cancer during their lifetime, with an individual's relative risk depending on many factors, including age, lifestyle and genetic make-up. Much research has gone into identifying the genes that are mutated in tumorigenesis with the overwhelming majority of genetically-modified (GM) mice in cancer research showing accelerated tumorigenesis or recapitulating key aspects of the tumorigenic process. Yet if six out of ten people will not develop some form of cancer during their lifetime, together with the fact that some cancer patients experience spontaneous regression/remission, it suggests there are ways of 'resisting' cancer. Indeed, there are wildtype, spontaneously-arising mutants and GM mice that show some form of 'resistance' to cancer. Identification of mice with increased resistance to cancer is a novel aspect of cancer research that is important in terms of providing both chemopreventative and therapeutic options. In this review we describe the different mouse lines that display a 'cancer resistance' phenotype and discuss the molecular basis of their resistance.

The tumor suppressor gene RASSF1A is inactivated through point mutation or promoter hypermethylation in many human cancers. In this study, we conducted a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Rassf1a-null mice to identify candidate genes that collaborate with loss of Rassf1a in tumorigenesis. We identified 10 genes, including the transcription factor Runx2, a transcriptional partner of Yes-associated protein (YAP1) that displays tumor suppressive activity through competing with the oncogenic TEA domain family of transcription factors (TEAD) for YAP1 association. While loss of RASSF1A promoted the formation of oncogenic YAP1-TEAD complexes, the combined loss of both RASSF1A and RUNX2 further increased YAP1-TEAD levels, showing that loss of RASSF1A, together with RUNX2, is consistent with the multistep model of tumorigenesis. Clinically, RUNX2 expression was frequently downregulated in various cancers, and reduced RUNX2 expression was associated with poor survival in patients with diffuse large B-cell or atypical Burkitt/Burkitt-like lymphomas. Interestingly, decreased expression levels of RASSF1 and RUNX2 were observed in both precursor T-cell acute lymphoblastic leukemia and colorectal cancer, further supporting the hypothesis that dual regulation of YAP1-TEAD promotes oncogenic activity. Together, our findings provide evidence that loss of RASSF1A expression switches YAP1 from a tumor suppressor to an oncogene through regulating its association with transcription factors, thereby suggesting a novel mechanism for RASSF1A-mediated tumor suppression.

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm) embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm) embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

Background: CADM1 encodes an immunoglobulin superfamily (IGSF) cell adhesion molecule. Inactivation of CADM1, either by promoter hypermethylation or loss of heterozygosity, has been reported in a wide variety of tumor types, thus it has been postulated as a tumor suppressor gene.

Findings: We show for the first time that Cadm1 homozygous null mice die significantly faster than wildtype controls due to the spontaneous development of tumors at an earlier age and an increased tumor incidence of predominantly lymphomas, but also some solid tumors. Tumorigenesis was accelerated after irradiation of Cadm1 mice, with the reduced latency in tumor formation suggesting there are genes that collaborate with loss of Cadm1 in tumorigenesis. To identify these co-operating genetic events, we performed a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Cadm1 mice, and identified several common insertion sites (CIS) found specifically on a Cadm1-null background (and not wildtype background).

Conclusion: We confirm that Cadm1 is indeed a bona fide tumor suppressor gene and provide new insights into genetic partners that co-operate in tumorigenesis when Cadm1-expression is lost.

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.

Chi Wong

- Postdoctoral Fellow

I am clinically trained haematologist with an interest in blood cell disorders. I undertook PhD training at the MRC Laboratory of Molecular Biology in Cambridge before joining the Sanger Institute.

Research

To understand the role of cancer gene aberrations discovered by genome sequencing efforts, I generate genetically tractable model systems to recapitulate these aberrations in vitro and in vivo. I study the phenotypic consequences of cancer-associated gene mutations using these models, which can also be used to investigate the underlying mechanisms underpinning the observed phenotypes. By understanding the mechanistic action of cancer-associated gene mutations, I hope to uncover new therapeutic strategies that can specifically target cellular pathways critical for cancer maintenence.

Robin van der Weide

- MSc. intern

Honours-student of the master "Cancer, Stem Cells and Developmental Biology" at Utrecht University. Interested in complex genetics and computational biology of vertebrate development and cancer. Previous work includes an internship in the group of Prof. Dr. Edwin Cuppen at the Hubrecht Institute.

Research

Studying predisposition for familial melanoma by functional annotation and analysis of SNPs from a large cohort.